The faithful segregation of genetic material to daughter cells is essential for the survival of an organism. The cell must replicate the DNA of each of its chromosomes and distribute one copy to each of the two daughter cells. Chromosome segregation is mediated by the mitotic spindle, which is composed of a dynamic array of microtubules and associated proteins. One of the most complex aspects of mitosis is the congression of chromosomes to the metaphase plate, which involves a multitude of forces acting on the kinetochore, on the chromosome arms, and on the spindle itself. Several models have been proposed for the molecular origins of these forces as well as for when and where they act on the spindle, but we do not have a clear understanding of how these forces are integrated within the spindle to achieve proper and timely chromosome alignment. In the present proposal we will: 1) Determine the temporal and spatial defects in chromosome positioning caused by perturbation of key molecules in the congression by knocking down molecules that we hypothesize contribute to different elements of chromosome congression and then performing a high-resolution fixed cell analysis in which we identify the locations of all chromosomes/ kinetochores in the spindle as well as measure the defects in the timing of mitotic progression, 2) Develop a new kinetochore tracking algorithm and associated data analysis to test the hypothesis that kinetochore movements during congression will have measurable characteristics that depend upon a specific molecular mechanism, and 3) We will ask how chromosome congression is spatially, molecularly and temporally controlled by applying the tracking algorithms developed in Aim 2 along with the molecular perturbations outlined in Aim 1 to quantitatively analyze pre-anaphase chromosome movement. We will perform a series of molecular perturbations that should disrupt one or more pathways of congression and then ask how chromosome movement is altered. Together these studies will provide significant insight into the factors that govern chromosome behavior as well as how chromosome behavior is spatially and temporally coordinated. PUBLIC HEALTH RELEVANCE: The faithful segregation of genetic material by the mitotic spindle to daughter cells is essential for the survival of an organism. The spindle is composed of microtubules and associated proteins that are utilized to attach the chromosomes to the spindle and to ensure their accurate segregation. Given that the mitotic spindle is a target of numerous chemotherapeutic agents that specifically disrupt spindle MT dynamics, elucidating the mechanisms by which spindle microtubule dynamics are regulated has important implications for biomedical research.